Translocations affecting a core binding factor (CBF) gene are one of the most frequent genetic aberrations in human acute myeloid leukemia (AML), accounting for up to 25% of all AMLs. Nearly half of these are associated with translocations affecting chromosome 16, resulting in expression of the fusion protein CBF(- MYH11. The overall objective of the proposed studies is to define the signaling pathways and downstream target genes that are affected by CBF(-MYH11, to evaluate the role of these regulated genes in leukemic stem cell self-renewal, survival and differentiation, and to identify the signaling pathways that cooperate with CBFP-MYH11 in leukemogenesis. To accomplish this goal, we propose to use the model of fusion gene expression in human CD34+ cells we have developed, to study the pre-leukemia that is a result of CBF(- MYH11 expression prior to the acquisition of cooperating mutations that lead to frank leukemia. We have successfully used this system to model the related leukemia fusion protein AML1-ETO. The specific aims of this proposal are as follows: Aim 1: Define the signaling pathways downstream of CBF(-MYH 11. Expression of CBF(-MYH11 in human CD34+ cells leads to their continued growth in vitro for an extended period of greater than five months. These cells will be characterized in terms of their proliferation, survival and differentiation, and compared to the long-term cultures initiated by the related CBF fusion, AML1-ETO. Aim 2: Identify target genes of CBFp-MYHl1 and analyze their role in CBF(-MYH11 -induced proliferation. A common gene expression profile will be identified by comparing the transcriptome of CD34+ cells from our long-term cultures with normal CD34+ cells. We will analyze the contribution of these regulated target genes by over-expressing those that are repressed, and conversely by knocking-down those genes that become upregulated. Aim 3: Identify the signaling pathways that cooperate with CBF(-MYHl1 in leukemogenesis. The pre-leukemic cultures expressing CBF(-MYH11 will serve as the background in which cooperating mutations will be tested. To identify specific signals that will cooperate in the transformation to acute leukemia, defined genetic elements and saturating retroviral mutagenesis will be used. [unreadable] [unreadable]